Hepatitis C virus is a major etiologic agent of hepatocellular carcinoma (HCC). HCV-induced HCC is typically preceded by chronic hepatitis and cirrhosis. Studies suggest that HCV has a direct oncogenic potential and increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage that can be mutagenic in the development of cancer. However, the source of reactive oxygen species (ROS) and how HCV induces HCC through oxidative stress, secondary to chronic inflammation/cirrhosis, are still largely unresolved. Recently, our laboratory discovered that hepatocyte NAD(P)H oxidases 1 and 4 (Nox1 and Nox4) are prominent sources of ROS in hepatocytes producing infectious HCV as well as in the HCV-infected human liver. Furthermore, Nox4 was prominent in the nucleus of these cells, particularly in the presence of HCV, and increased the generation of ROS and nitrotyrosine in the nucleus which is likely to increase the probability of reactive species damaging the host nuclear DNA in carcinogenesis. HCV elevated Nox4 through a fibrogenic cytokine, transforming growth factor beta 1. In addition, HCV infection is characterized by chronic, low level of inflammation. Toll-like receptor 4 (TLR4) and tumor necrosis factor alpha (TNF?) are elevated in hepatitis C patients. Nox4 is required for TLR4 signaling, and TNF? can induce Nox4. Thus, hepatocyte Nox4 may serve as a link between inflammation, fibrosis, and cancer during HCV infection. Importantly, the ability to regulate Nox enzymes makes them potential targets for therapy. Nevertheless, the pathogenic consequence of persistent Nox4 induction by HCV and combined oncogenic effects of HCV plus inflammation have not been clearly delineated. Therefore, the goal of this study is to determine the role of Nox enzymes in the HCV- and inflammation-induced DNA damage in hepatocarcinogenesis, focusing on hepatocyte Nox4. The study will employ infectious HCV-producing cell culture systems and an HCV animal model. We hypothesize that hepatocyte Nox4, together with inflammation-driven Noxes and other sources of ROS, increases mutations to the host DNA to facilitate the development of HCC during chronic HCV infection. The specific aims are: 1) to determine the role of hepatocyte Nox1 and Nox4 in the HCV-induced oxidative modification/mutations to the host DNA in hepatocarcinogenesis. Subcellular localization of Nox1 and Nox4 and molecular determinants of perinuclear/nuclear location of Nox4 will continue to be characterized as they relate to the probability of reactive species damaging the host DNA; 2) to determine the role of Nox4 in the spontaneous, HCV-induced hepatic tumors in vivo. HCV transgenic/Nox4 knockout mice will be generated by crossing HCV core transgenic mice with Nox4 knockout mice, which are available, to examine whether the rate of DNA damage and hepatic tumors decreases in the HCV transgenic mice with genetic ablation of nox4 gene; 3) to examine the combined effects of HCV and inflammation on the hepatocyte Nox enzymes, DNA damage, and hepatocarcinogenesis. Results from this study will increase our understanding of hepatocyte Nox4 and the role of inflammation in cancer, for the development of therapy. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a growing public health concern and is estimated to have infected nearly four to five million individuals in the U.S. alone. One of the most devastating consequences of HCV infection is hepatocellular carcinoma, which is still on the rise. This study examines newly described hepatocyte NAD(P)H oxidases (Nox proteins) as a persistent and robust, intra/peri-nuclear source of reactive species, which would promote the DNA damage in response to fibrogenic and pro-inflammatory cytokines increased by HCV, thereby linking chronic hepatitis/fibrogenesis to hepatocarcinogenesis during HCV infection. Results from this study will increase our understanding of HCV, Nox proteins, and the relationship between oxidative stress and HCV-induced liver disease for the development of therapy directed at the source of the reactive species.